Use of Baculoviruses as Biological Insecticides

Transcription

1 Use of Baculoviruses as Biological Insecticides Jenny S. Cory* and David H. L. Bishop Abstract Naturat!y!occurring baculoviruses can ~ =used to controi a wide range of insect ~'sts. Most baculoviruses are ~ed, las biopesticides, that is, they are sprayed onto high-density pestpopula~0ns inamanner akin to the use 0f~ynthetic clieiriical pesticides. Howeve~, Other strategies that use the biological features 0f the:v~ses ~e at~ol possible~::and Shouid increase as we e~pand our knowledge of baculovirus e~oio~ii'j~n~ order to fi0n systems todetailed study of pest behavior and the development of appropriate aoplicafion strategies. lrtdex Enti'ies: Biopesticides; pest control; inseet viruses; bioassay; production; app!igati0n. 1. Introduction Baculoviruses have been isolated from a wide range of invertebrates. Their development as pest control agents spans over 40 years, although records of their discovery and use date from considerably earlier (1). The baculoviruses from Lepidoptera (butterflies and moths) and Hymenoptera (sawflies only), and the now unclassified nonoccluded viruses from Coleoptera (beetles) present the best options for pest control, and thus most research on baculovirus insecticides has concentrated on these orders. Baculovirus insecticides have been used in a wide range of situations from forests and fields to food stores and greenhouses. Baculoviruses have several advantages over conventional insecticides that make them highly acceptable control agents. Probably the most important is their specificity. They have narrow host ranges (sometimes limited to one or two species), and do not infect beneficial insects, making them very suitable for use in integrated control programs. Most also possess the capacity to persist in the environment, which can be utilized in the development of more ecologically based long- term control programs. Baculoviruses are particularly suitable for use in developing countries, since they can be produced locally and their production in vivo (although labor-intensive) requires little in the way of capital expenditure. More recently, interest in baculoviruses has expanded in relation to their potential for genetic modification, primarily to increase their efficacy. This area is still in its infancy, and the development of genetically modified baculoviruses will not be considered here (however, see Cory [21, Possee et al. [31, and Cory et al. [3a1), although most factors that are relevant to their use in the field will be the same as for naturally occurring baculoviruses. The use of baculoviruses as biological insecticides is a broad subject area. The rationale taken in this article has been to outline the pathway required for the development of a control program utilizing baculoviruses, from isolation to field trials, and also to describe aspects of the theoretical background (see Fig. 1). The further development of baculoviruses for use in integrated control programs and their commercialization are beyond the scope of this article. *Author to whom all correspondence and reprint requests should be addressed. Centre for Ecology and Hydrology, Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford 0X1 3SR, UK, JSC@mail.nerc-oxford.ac.uk Molecular Biotechnology 9 Humana Press Inc. All rights of any nature whatsoever reserved /7:3/ /510.75

2 I Preliminary [ [Laboratory[ Larger scale laboratory testing I development field testing Phase I Phase II Phase IV J ] Integrated field ] trials l [ C~176 1 Phase V Phase V[ [-~l Development of [ optimal virus I"1 l production I Development of ] systems I ] methodof Isolation and / \ apphcatton biochemical[ /F-if[ \ / I characterization / /t...j \ / (, I I Formulation I\ I ~ / /I devel~ [,~\ / [~ Appllcatioo //A I strategy / \ Prelimlnary I / \ {I ~ [ field testing i / Laboratory I \ ' ' / \ I efficacy testing ] \ / \ Pest biology and [ behaviour / Phase III [ Field studies and I small scale trials Formulation testing Interaction with other control agents Safety testing I Registration I Fig. 1. Schematic pathway for the development of baculoviruses as control agents. Numbered boxes refer to those categories dealt with in the text. 2. Materials The majority of baculoviruses have been isolated from Lepidoptera: over 500 to date (4). Whether this reflects a genuine host bias or is merely a product of the fact that there are large numbers of lepidopterists (compared to other insect specialists) and that baculovirus infected larvae are usually very obvious, remains to be seen. However, it does indicate that for Lepidoptera at least, there is a high probability of isolating a baculovirus from a given species. Many pest-control programs rely on the isolation of local strains of baculoviruses. This can have some advantages: it will circumvent any problems that may occur if an "exotic" virus is imported for wide-scale release, and may result in the selection of a virus better adapted to a particular host or ecosystem. Isolation of wild-type baculoviruses from insect pests can vary in difficulty. In families, such as the Lymantriid moths (for example, the gypsy moth, Lymantria dispar, and the Douglas fir tus- sock moth, Orgyia pseudosugata), virus epizootics are common and isolates are not difficult to collect, particularly in high-density populations. The apparent rarity of epizootics in some other species, however, does not preclude the successful use of a baculovirus for their control. The current means of characterizing a newly isolated baculovirus is by restriction endonuclease digestion of its genomic DNA. The resulting DNA profile gives a good indication of the uniqueness (or otherwise) of the isolate. It is also highly likely that any wild-type isolate will be a mixture of several genotypically distinct varieties, for example, the lepidopteran Helicoverpa spp. nuclear polyhedrosis viruses (NPVs) (5) and the stem borer Chilo spp. granulosis viruses (GVs) (6). The relevance of this heterogeneity to successful pest control has yet to be ascertained. 3. Methods Every pest:crop system will present its own unique problems, but the general approach and

3 areas for study will be similar for most pest control programs. The most important of these areas are discussed in the following sections Screening and Efficacy The first step in the initiation of any pestcontrol program using baculoviruses is the screening of available isolates to assess their efficacy as potential control agents. This can only be accomplished by insect bioassay. Production and registration of baculoviruses also require the establishment of a standard bioassay system. Bioassay of baculoviruses basically describes the relationship between the virus and the insect in terms of overall mortality or rate of kill. The standard dose-response curve of insects to baculoviruses is sigmoidal. When setting up a bioassay, a range finding assay should be carried out first, using a large range of doses, to assess the shape of the response and to pinpoint the linear region of the curve. A second assay is then set up with the doses arranged symmetrically around the lethal concentration (LC)50 or lethal dose (LD)5o point. The spacing of the doses and the number of test insects can vary, but in general, if the number of doses (and subjects) is low, the doses should be widely spaced and vice versa. Several parameters can be measured using the bioassay: The LCso and LDs0 are the most commonly used in dose-mortality studies. The LCs0 is the concentration of virus that the test insects feed on or drink that results in 50% being killed. An LDs0 is the dose at which there is 50% mortality of the test insects. An LCs0 tells you less about the response than the LDs0, since in the former, the amount of virus ingested is dependent on the behavior of the insect and so is inherently more likely to vary between tests, whereas the LDs0 is independent of differences in individual behavior patterns, making this technique more suitable for comparative experiments. The most common method of analysis for dose-mortality data is probit analysis, which is used to determine the LDs0 and LCs0 values, although other models are also used (7,8). Analysis of the time-mortality response (time to death for 50% of the test insects) can be mea- sured either as the lethal time (LT)50, where test insects are continually exposed to virus, or as the survival time (ST)50, where the inoculum is only received at the beginning of the assay. Probit analysis is not valid for analyzing time-mortality assays, and other transforms, such as the logit, should be used for these data. Many factors will affect the outcome of bioassays, including the instar used, temperature, the choice of insect diet, and larval weight. In order to increase the precision of the assay, it is important to minimize heterogeneity within the system, in particular, by using test insects within a very narrow weight range. The assay must be standardized, particularly if batches of virus are being tested against a standard, as in a potency assay. Several insect bioassay techniques have been developed, and there are numerous variants of these to account for the different feeding requirements of particular insect species. It is not possible to make valid comparisons between different bioassay techniques, since each will generate different values. The two most commonly used bioassays are described here I. Diet Plug Assay Many Lepidoptera, and in particular, noctuid species, will feed on an artificial or semiartificial diet, which makes insect bioassays much easier to handle than when foliage is used. Details of a range of diets and the insects that have successfully been reared on them can be found in Singh and Moore (9). 1. Place a small plug of diet (of a size suitable for the chosen instar to eat within 24 h) in the base of a 96-well microtiter plate (a larger plate will be needed for the larger instars). 2. Apply 1 IxL of the virus suspension to the diet plug with a micropipet. 3. Place larvae individually in wells and cover. It is usually best to seal the microtiter plate with damp tissues to prevent dehydration of larvae or diet, and to reduce the likelihood of escape. 4. Place the microtiter plate at constant temperature for 24 h. Transfer larvae to small individual pots that contain enough diet to maintain them through to pupation. Only transfer those insects

4 that have completely consumed their diet plug and have therefore taken up a measured dose of virus. Seal and perforate the lids. 5. Check after 24 h to remove handling deaths, and then monitor and record daily until death or pupation. 6. With well-infected larvae, diagnosis can often be made visually. However, with early deaths or small larvae, it is often difficult to do this. The easiest method of confirming diagnosis of NPVs is to make a thin smear of the midgut region, stain with Giemsa, and observe at xl000 under oil immersion. Granulosis viruses will need further confirmation using alternative techniques, such as ELISA, dot blotting, or by the transmission electron microscope Droplet Feeding Assay This particular system was developed by Hughes and Wood (10) and has since undergone numerous improvements (8). The technique was developed for neonates, however, it has also been successfully used for later instars (loa). Its main advantage is that the dose is ingested over a very short time, and infection can thus be regarded as synchronous. This is of particular importance in the measurement of STs0 responses. 1. Preparation and selection of the larvae: Larvae are chosen from a narrow window of hatch time, 3-5-h old. They are then selected for vigor by allowing them to climb a ramp of blotting paper. 2. Larvae are placed on a plastic surface that has been ringed with a 2:1 mix of mineral oil and vaseline to prevent them from escaping. The virus suspension is then placed in small drops near the larvae. The suspension should contain a small amount of blue food coloring. 3. Larvae that have fed on the virus solution should be obvious because of the blue dye in their gut. These larvae are transferred carefully to individual pots containing diet. 4. Monitor as appropriate. For LTs0 or STs0 analysis, this may well need to be every few hours. The bioassay carried out in this manner can be used to estimate the LCs0. The amount of fluid ingested (and thereby the dose) needs to be estimated for evaluation of an LDs0. This can be done using 32p (10), by fluorescence spectroscopy (11), or gravimetrically Virus Production At present, natural viruses for pest control are produced in vivo. The use of an in vitro system for virus production is some time away, because the yield of virus/unit volume is still too low to be considered economically feasible and the virus often loses infectivity for the insect host after passage through cell culture. With larger-scale production programs such as for the corn earworm Helicoverpa zea NPV (12) and gypsy moth, L. dispar NPV (13,14), the factors affecting the optimization and cost effectiveness of in vivo virus production have been studied in more detail. The most important aspects of the production process are: choice of host, dose/instar relationship, rearing conditions, and virus processing, all of which are outlined in Subheadings For a more detailed discussion of these and other areas, see Shapiro (14) and Carter (15) Choice of Host It is usually preferable to use the natural virus host, which, in order to reduce the likelihood of contamination, should be reared in the laboratory rather than collected from the field. It is also a considerable advantage to use a host species that has been adapted to an artificial diet. Alternative hosts must be sought if the natural host is unsuitable for laboratory culture, for example, where the dietary requirements are too stringent, where a long obligatory diapause is required, or if it produces irritant, urticacious hairs. In these circumstances, it is important to confirm the identity of the progeny virus, since alternative hosts might select other strains from the original (mixed) inoculum or switch to a homologous virus if this is present in a latent state within the alternative host culture Instar-Dose Relationship The aim of the production process is to use the minimum quantity of inoculum to produce the maximum yield and quality of virus. This will require several range-finding experiments in order to optimize the process. For instance, Kelly and

5 Entwistle (16) compared the use of third, fourth, and fifth instar cabbage moth, Mamestra brassicae larvae for NPV production. Third instars clearly gave the larger yield, which did not vary within the dose range tested (6.25 x x 108 polyhedral inclusion bodies [PIBs]/mL). Other factors can also affect the viral product. For example, activity of virus appears to vary depending on when it is harvested from the larvae. NPV collected from H. zea larvae was found to be more active and more abundant when it was recovered from dead larvae rather than from live ones (12). Similarly, in L. dispar, the activity of the NPV obtained from different instars varied; it was found to increase up to the fourth instar and then decrease in the fifth (17) Rearing Conditions In general, the most important environmental consideration is temperature. Other factors, such as humidity, appear to have little affect on virus production, except perhaps in terms of creating favorable conditions for the growth of undesirable contaminants. The optimum temperature range appears to be between 20 ~ and 26~ Shapiro (14) investigated gypsy moth, L. dispar, NPV production at temperatures between 23 ~ and 32~ and found that at the highest temperatures, both yield and activity of the NPV were lower. Similarly, Kelly and Entwistle (16) looked at the effect of temperature on the rearing of both M. brassicae and the pine beauty moth, Panolisflammea NPVs in M. brassicae larvae over the range of 20 ~ to 30~ The results again tended to point to a reduction in yield with an increase in temperature. The other key factors for virus production are size of container and the number of larvae reared/ container. Harvesting the virus-infected larvae is the most time-consuming, and thus expensive, stage of the virus production process, so it pays to use larger containers, although this must be set against the cannibalistic nature of many species Processing The processing of virus-killed larvae and the level of purification used greatly affect the final yield. To liberate the virus from the larval bodies, the insects are blended with diluent and then filtered through muslin. Both the ratio of body weight to diluent and time spent blending have a significant affect on the final virus yield. Shapiro (14) showed with L. dispar NPV that using 1 g larval weight with 10 ml distilled water resulted in 95% PIB recovery after filtration. At lower dilutions, a greater proportion of the virus was lost. Similarly, with blending time, 5 s in a blender resulted in 75% PIB recovery, whereas 15 s resulted in 95%. A comparison of semipurification and full purification has been made for the production of M. brassicae and the brown tail moth, Euproctis chrysorrhoea, NPVs (16,18). Semipurification involved blending and coarse filtration, followed by low-speed contrifugation to remove debris and a higher-speed spin to pellet the virus. Both the low- and high-speed spins were repeated to ensure maximum virus recovery. For full purification, these steps were followed by centrifugation at 30,000g through a discontinuous 50-60% sucrose gradient. With M. brassicae NPV, the fully purified product resulted in a loss of 25-30% of the virus and E. chrysorrhoea NPV with a 35% loss of PIBs as compared to the semipure preparation. The authors calculated that the ratio of PIBs produced to inoculum used was >1000:1 for E. chrysorrhoea NPV, 2200:1 for M. brassicae NPV, 2150:1 for O. pseudosugata NPV, and 4000:1 for L. dispar NPV Formulation The formulation of baculoviruses, although important, has not really received much attention. Formulation of baculovirus insecticides basically falls into two areas: that relating to storage stability and factors important to field application. Storage stability is very much dependent on the method of processing, but it has been little tested and there are few guidelines on it. The main aims are to produce a stable preparation in which the viability of the baculovirus is preserved or even enhanced. Most baculoviruses are processed for use as sprays, and research into the production of solid preparations is much rarer. For small-scale trials, storage stability is not usually a problem

6 since macerated larvae mixed with water or partially purified preparations are often very effective in the field and will keep reasonably well for short periods provided they are refrigerated or, even better, frozen. This is not as practical for larger quantifies of virus, where there is a need to move away from time-consuming steps, such as centrifugation, and where it is important to keep contaminant levels low. Most baculovirus products are produced as concentrated wettable powders. The main methods for large-scale processing and formulation are spray- or air-drying after dilution with an inert carrier (e.g., the commercial H. zea NPV product "Elcar"), the more expensive process of freeze-drying (lyophilization) with a carbohydrate, often lactose (e.g., US Forest Service formulations ofl. dispar NPV and O. pseudosugata NPV), or acetone precipitation in lactose. In terms of stability, the indications are that spraydrying produces the best quality product, although freeze-drying powders also retain activity. Preparations produced by acetone precipitation are not favored, because they tend to lose activity in storage. In general, NPVs appear to be more stable than GVs, and aqueous preparations lose activity more rapidly than dry formulations. For further details, see reviews by Couch and Ignoffo (19) and Young and Yearian (20). Formulation for the field must provide good residual activity on the target site, and is thus dependent on the nature of the substrate and the effect of environmental parameters (temperature, humidity, sunlight, and so on). Little is actually known about the effect of substrate on virus viability, but of the environmental parameters that might affect them, only ultraviolet (UV) radiation between 290 and 320 nm is thought to have any major influence in inactivating the virus. Baculoviruses completely lose activity in a matter of hours in direct sunlight. The addition of UV protectants and other compounds to baculovirus formulations have been rather random and unsystematic in terms of their assessment. In general, the types of compounds that have been added to baculovirus formulations to act as UV protectants fall into two categories: UV reflectants and UV absorbers. The former are primarily metallic oxides, and the latter are a varied mixture of substances that include antievaporants (to delay droplet evaporation during spraying), feeding stimulants, spreaders/wetting agents (usually detergents added to reduce aggregation), and stickers (although adhesion to many plant surfaces appears to be very strong) (1). More recent research has also shown that some optical brighteners such as Tinopal, can enhance activity by up to 1000-fold (20a). Whatever additives are included in baculovirus formulations, care must be taken to check that they have no detrimental effects on virus activity. Since baculoviruses break down in alkaline conditions, ph is obviously particularly important. Additives must also be tested to see that they do not encourage aggregation or reduce adhesion of PIBs to the target substrate. They must also be compatible with the spray equipment and, where applicable, with other pesticides in the final tank mix Pest Biology The two most important aspects of baculovirus application are temporal and spatial. However effective a virus isolate is in the laboratory, it will fail in the field if it is not applied in the right place and at the right time. Factors relating to pest biology and behavior can be crucial to the success of pest-control programs. Knowledge of insect behavior on the crop after hatch, its distribution within the crop canopy in each instar and the area of foliage ingested per instar will allow precise and effective use of viral pathogens. The optimal quantity of virus required per unit area of host plant can be estimated and used together with the behavioral information to select the most appropriate application methodology and strategy (see Subheadings 3.5. and 3.6.). For Lepidopteran pests, the damage is usually done in the larval stage, with most being carried out in the final two instars. To minimize crop damage, the baculovirus must be applied as early as possible so that death occurs before the insect reaches the final instars. If the pest is exposed continually during its life cycle and is thus accessible to the baculovirus spray, then the development of the control program does not usually present a problem. However, many pests

7 are protected from insecticidal sprays at some time during their life cycles, for example, stem borers, leaf curlers, and soil-dwelling cutworms, which demands that both timing and deposition of virus be very accurate so that the insect ingests a lethal dose of virus before it enters its refuge. In some cases, this may necessitate the development of specialized application techniques Method of Application Baculoviruses need to be ingested by the insect in order to initiate infection. Thus effective application should distribute virus to the pest's feeding sites and give good coverage, so that the insect's opportunity for acquiring a lethal dose of virus is maximized. Most baculoviruses are applied as sprays, which has meant that they can be, and are, applied using the same equipment as conventional pesticides. In forests, the normal method for application is from the air using airplanes or helicopters fitted with either hydraulic nozzles on a boom or spinning-disk atomizers. For agricultural crops, most spraying is carried out from the ground using boom and nozzle equipment with either fiat-fan or hollow-cone jets. In orchards, plantation crops, and some specialized situations, such as in nature reserves or roadside verges, small-scale application equipment, such as back-pack mist blowers and hand-held sprayers with either hydraulic nozzles or spinning-disk applicators, can be useful. Electrostatic sprayers have received little attention with regard to the application of microbial pesticides, but they have some disadvantages since canopy penetration is not usually very deep. Because it is possible to apply baculoviruses using the aforementioned techniques, there is a tendency to use whatever equipment is available, rather than determining what application criteria are most suitable for the particular crop-pest situation. Failure to obtain adequate viral infection in the field may be the result of ineffectual application, rather than innate problems of the host-virus system itself. Judicious timing of application is part of this process, since larger quantities of virus are needed as the larvae age and decrease in susceptibility, for example, as has been studied in detail for the pine beauty moth, P. flammea (21). In this example, it was found that prehatch virus application was ineffective as compared to posthatch sprays. There was only a small window for application after hatch if virus mortality was to be high and crop damage avoided. The optimal droplet size, droplet density, and dose/drop should be estimated for each particular system, and then the spray equipment selected to deliver it. A key factor in application seems to be spray droplet size. With chemical sprays, small droplets ( ~n) give better surface coverage on foliage with a resulting increase in pest mortality (22). This has also been found to be the case with many baculoviruses, presumably because it increases the chances of the insect encountering a PIB-containing droplet, although there are some notable exceptions to this, such as with Helicoverpa control on cotton (1). There is a move toward controlled droplet application using low-volume and ultralowvolume equipment, such as spinning-disk sprayers, which are now commonly used for the application of baculoviruses in forests. It is particularly important to keep tank volume down in the forest situation because of the high costs of aerial spraying and the logistic difficulties of carrying large quantities of fluid to the site. The depth of the crop in forests can also be very large. Thus it is important to consider and measure canopy penetration in relation to the distribution of insects on the crops when deciding on suitable application systems. For some groups of pests, standard spray application is not going to be effective or is undesirable for other reasons. One such example is the group of noctuid pests known as cutworms. Because these insects spend most of their lives hidden in the soil, normal spray application is not particularly effective, and such alternatives as development of baits, usually based on bran, are being investigated instead (22a). Stored product pests represent another special case: water-based sprays may encourage the growth of fungal agents in stores. Thus techniques using dusts are being studied for delivery of viruses to stored product pests.

8 Other application methodologies include the release of infected insects, a technique that has been particularly successful with nonpersistent nonoccluded viruses (see Subheading ). Further novel suggestions include luring adults to light or pheromone traps, where they become covered in a baculovirus dust, which is transferred on to their eggs, and subsequently infecting the crop of parasitoid wasps that have been contaminated with virus solution, who will act as vectors for the baculovirus Application Strategy The majority of baculovirus applications fall into one category, inundative release, and they are therefore in direct competition with chemical pesticides. Other more long-term control strategies have been investigated and are discussed in the following sections. In general, ecologically based control strategies have received little attention, since they require a good understanding of the long-term effects of baculoviruses. The use of baculoviruses as classical biological control agents (where introduction of an exotic virus results in self-sustaining control) is not likely to be possible in many circumstances. This is primarily because in most habitats, the virus will be rapidly removed from the host environment. Virus is only likely to persist in stable crop systems and where there is an element of vertical transmission via the host or alternative hosts. The only example with baculoviruses that can be said to fall into this category is the outbreak of an NPV in the spruce sawfly, Gilpinia bercyniae, in Canada, where an NPV was accidentally introduced into the population (23). The population has not resurged since the viral epizootic, a period of almost 50 yr. Application of baculoviruses at more strategic times to slow down the pest population build-up is a more likely option, and may be a more effective and less costly alternative to other control methods in more stable habitats. Early spraying (i.e., preoutbreak) is now the recommended strategy for Douglas fir tussock moth, O. pseudosugata, control in North America. The main strategies for applying baculoviruses fall into three categories: 1. Inundative release; 2. Inoculation; and 3. Manipulation of resources Inundative Release This strategy involves the application of a very large quantity of virus when the pest has reached outbreak proportions, with the expectation that the control will only be of limited duration. Currently this is by far the most common approach to applying baculoviruses in both forest and agricultural situations. In forests where single, univoltine pests are often the target, one application is usually sufficient. In agricultural crops, where pest complexes are more common and multiple generations can occur throughout the year, applications will have to be more frequent. Alternatives to blanket, high-dose coverage have been suggested, for example, more frequent applications of lower-dose sprays in agricultural situations, but these have been little studied so far. Another suggestion is that of lattice introductions, which makes use of the dispersive capacity of the virus. With information on the rate of dispersal in a specific virus:host ecosystem, the virus could be introduced in strips from which it would spread to the intermediate areas of infestation, thus saving both virus and flying time Inoculation This approach is based on long-term regulation where it is anticipated that the effect will not be permanent and will require reintroduction of virus at a later stage. Virus is introduced into the population at low levels with the intention that it will build up within the population over a number of generations and suppress pest numbers. The most successful example of this strategy is control of the rhinoceros beetle, Oryctes rhinoceros in the Pacific region. The virus, which is used to control this pest, used to be classified as a subgroup C baculovirus, but has since been reclassified as a nonoccluded virus. Since representatives of this group have no protein coat, they have limited persistence in the environment and are not suitable for spray application. However, they can be very effectively introduced into the environment by releasing infected hosts. Infected adult beetles

9 continually excrete virus and in this way contaminate feeding and breeding sites, thereby passing on the infection to both adults and larvae. The virus can also be transmitted during mating and in the females this severely reduces fecundity (24) Manipulation of Resources The natural body of baculovirus inoculum in the environment is manipulated in such a way that it is brought into repeated contact with the pest and so maintains numbers at economically acceptable levels. This strategy is only suitable in more permanent habitats and makes use of the persistent capacity of the virus. This technique requires an intimate knowledge of the dynamics of the baculovirus:host relationship and has as yet only been applied successfully to the lepidopterous pasture pests Wiseana spp. in New Zealand. Where the pasture was more frequently ploughed, pest outbreaks were more common because any virus remaining in the soil was rapidly removed from the pest environment. By instigating a minimal disturbance regime, the virus pool could be manipulated so that it was available to infect Wiseana, and in this way, the pest was kept below economically damaging levels (25). 4. Results Although baculoviruses have been isolated from several orders of insects, they have only been used to control pest species from two of them: Lepidoptera (from which the majority of baculovirus isolations have been made) and Hymenoptera (diprionid sawflies). Control of the sawflies is particularly successful because of their high degree of sensitivity, the gregarious nature of many species, and the presence of vertical transmission (26). Only one virus has been developed for the control of Coleopteran pests, that of the rhinoceros beetle, O. rhinoceros (see Subheading ). This virus has been used very successfully to control Oryctes spp. in coconut plantations in the Pacific regions and elsewhere, and its use may well be extended in order to control other susceptible dynastid beetles. Overall, the most successful examples of baculovirus usage are to be found in the control of forest pests, a fact that is mainly related to the higher damage threshold in forests. The development of baculovirus against forest pests is frequently undertaken by government agencies. For instance, in the United States and Canada, the forest services have registered baculoviruses for use against the Douglas fir tussock moth, O. pseudosugata, gypsy moth, L. dispar, pine sawfly, Neodiprion sertifer, and the red headed sawfly, Neodiprion lecontei, all of which have been used successfully in largescale control campaigns (27). However, the largest potential market for baculoviruses is in the agricultural sector. In recent years, several small companies have started to produce baculovirus insecticides, but they have yet to take over a significant share of the market. The first baculovirus to be commercialized was the NPV from the corn earworm, H. zea, for use on cotton. After a promising start, the sales of this product fell, primarily because of the increase in use of synthetic pyrethroids among cotton growers. With the development of resistance of pyrethroids, Helicoverpa NPV may again have a role to play in control of this pest. Other baculoviruses that have been looked at in terms of commercial development are the GV of the codling moth, Cydia pomonella, various Spodoptera NPVs, and the broad host range alfalfa moth, Autographa californica, NPV (27). Many other baculoviruses are being developed on a more local level. A particularly successful program is being carried out in Brazil, where NPV is being used over hundreds of thousands of hectares for the control of the velvet bean caterpillar, Anticarsia gemmatalis on soybean. The Chinese have made large collections of native isolates and are developing many of them for control of agricultural pests. Russia has also developed the production of several viruses, mainly for forest pests, to commercial levels. For more information on the use of baculoviruses as control agents, see reviews by Young and Yearian (28), Entwistle and Evans (1), and Huber (29). However, despite the obvious potential of baculoviruses for the control of many agricultural pests, their use is still limited. A key factor in this is their speed of action; because the viral infection necessarily takes time to develop, some feed-

10 ing damage can occur and in many agricultural crops this is, at present, unacceptable. The issue is more complex, however. Chemical insecticides are easy to use, and result in a rapid and obvious knock-down effect. Baculoviruses require more care and effort in application, including monitoring of the pest populations, and do not give such a dramatic affect. The expectations of farmers need to be changed before they gain acceptance. In terms of commercialization, their narrow host range is seen as a drawback. Equally, the likelihood of developing more ecologically based, long-term control strategies that utilize the persistent effect is also not likely to appeal to the more commercially minded manufacturers of pesticides. Thus, investment in baculoviruses is not as great as for other control methods, and their development is primarily left to publicly funded institutions. However, the current awareness of the dangers of using broad-spectrum, persistent chemical pesticides and the problems of pesticide residues in foodstuffs, together with the general move towards more environmentally friendly methods of pest control may well result in recognition of the great potential of baculoviruses, and we may see a more serious development of natural baculovirus insecticides in agriculture. References 1. Entwistle, P. F. and Evans, H. F. (1985) Viral control, in Comprehensive Insect Physiology, Biochemistry and Pharmacology (Kerkut, G. A. and Gilbert, L. I., eds.), Pergamon, Oxford, UK, pp Cory, J. S. (1991) Release of genetically modified viruses. Rev. Med. Virol. 1, Possee, R. D., King, L. A., Weitzman, M. D., Mass, S. D., Hughes, D. S., Cameron, I. R., Hirst, M. L., and Bishop, D. H. L. (1992) Progress in the genetic modification and field-release of baculovirus insecticides. Proceedings of Second International Conference on the Release of Genetically-Engineered Microorganisms (REGEM 2), Plenum, New York, pp a. Cory, J. S., Hirst, M. L., Williams, T., Hails, R. S., Goulson, D., Green, B. M., Caley, T. M., Possee, R. D., Cayley, P. J., and Bishop, D. H. L. (1994) Field trial of a genetically improved baculovirus insecticide. Nature 37, Martigoni, M. E. and Iwai, P. J. (1986) A Catalog of Viral Diseases of Insects, Mites and Ticks. 4th ed. USDA, Forest Service, General Technical Report, PNW Gettig, R. R. and McCarthy, W. J. (1982) Genotypic variation among wild isolated of Heliothis spp. nuclear polyhedrosis viruses from different geographic regions. Virology 117, Easwaramoorthy, S. and Cory, J. S. (1990) Characterization of the DNA of granulosis viruses isolated from two closely related moths, Chilo infuscatellus and C. sacchriphagus indicus. Arch. Virol. 110, Finney, D. J. (1978) Statistical Methods in Biological Assay. 3rd ed. Charles Griffin, London. 8. Hughes, P. R. and Wood, H. A. (1986) In vivo and in vitro bioassay methods for baculoviruses, in The Biology ofbaculoviruses, vol. II (Granados, R. R. and Federici, B., eds.), CRC, Boca Raton, FL, pp Singh, P. and Moore, R. F. (1985) Handbook of Insect Rearing, vols. I and II. Elsevier, Amsterdam. 10. Hughes, P. R. and Wood, H. A. (1981) A synchronous peroral technique for the bioassay of insect viruses. J. Invert. Pathol. 37, a. Smits, P. H. and Vlak, J. M. (1988) Biological activity of Spodoptera exigua nuclear polyhedrosis virus against S. exigua larvae. J. Invert. Pathol. 51, van Beek, N. A. M. and Hughes, P. R. (1986) Determination of fluorescence spectroscopy of the volume ingested by neonate lepidopterous larvae. J. Invert. Pathol. 48, I. 12. Ignoffo, C. M. and Couch, T. L. (1981) The nucleopolyhedrosis virus of Heliothis species, in Microbial Control of Pests and Plant Diseases (Burges, H. D., ed.), Academic, London, pp Lewis, F. B (1981) Control of the gypsy moth by a baculovirus, in Microbial Control of Pests and Plant Diseases (Burges, H. D., ed.), Academic, London, pp Shapiro, M. (1986) In vivo production of baculoviruses, in The Biology of Baculoviruses, vol. II (Ganados, R. R. and Federici. B., eds.), CRC, Boca Raton, FL, pp Carter, J. B. (1989) Viruses as pest control agents, in Management and Control of lnvertebrate Crop Pests (Russell, G. E., ed.), Intercept Ltd., Andover, UK, pp Kelly, P. M. and Entwistle, P. F. (1988) In vivo mass production in the cabbage moth (Mamestra brassicae) of a heterologous (Panolis) and a homologous (Mamestra) nuclear polyhedrosis virus. J. Virol. Methods 19, Shapiro, M., Robertson, J. L., and Bell, R. A. (1986) Quantitative and qualitative differences in gypsy moth (Lepidoptera: Lymantriidae) nucleo-polyhedrosis virus produced in different aged larvae. J. Econ. Entomol. 79, Kelly, P. M., Speight, M. R., and Entwistle, P. F. (1989) Mass production of Euproctis chrysorrhoea (L.) nuclear polyhedrosis virus J. Virol. Methods 25,

11 19. Couch, T. L. and Ignoffo, C. M. (1981) Formulation of insect pathogens, in Microbial Control of Pests and Plant Diseases (Burges, H. D., ed.), Academic, London, pp Young, S. Y., III and Yearian, W. C. (1986) Formulation and application of baculoviruses, in The Biology ofbaculoviruses, vol. II (Granados, R. R. and Federici, B., eds.), CRC, Boca Raton, FL, pp a. Shapiro, M. and Robertson, J. L. (1992) Enhancement of baculovirus activity of gypsy moth (Lepidoptera Lymantriidae) by optical brighteners. J. Economic Entomol. 85, Cory, J. S. and Entwistle, P. F. (1990) The effect of time of spray application on infection of the pine beauty moth, Panolis flammea (Den and Schiff) (Lepidoptera: Noctuidae) with nuclear polyhedrosis virus. J. Appl. Entomol. 110, Matthews, G. A. (1979) Pesticide Application Methods, Longman, London. 22a. Bourner, T. C., Vargas-Osuna, E., Williams, T., Santiago-Lavarez, F., and Cory, J. S. (1992) A comparison of the efficacy of nuclear polyhedrosis and granulotis viruses in spray and bait formulations for the control ofagro segetum (Lepidoptera: Noctuidae) in maize. Biocontrol Sci. Technol. 2, Neilsen, M. M., Martineau, R., and Rose A. H. (1971) Diprion hercyniae (Hartig), European spruce sawfly (Hymenoptera: Dipriondae); Biological control programs in Canada Tech. Bull. No 4 Commonw. Inst. Biol. Control Bedford, G. O. (1981) Control of the rhinoceros beetle by baculovirus, in Microbial Control of Pest and Plant Diseases (Burges, H. D., ed.), Academic, London, pp Kalmakoff, J. and Crawford, A. M. (1982) Enzootic virus control of Wiseana spp. in a pasture environment, in Microbial and Viral Pesticides (Kurstak, E., ed.), Dekker, New York, pp Cunningham, J. C. and Entwistle, P. F., (1981) Control of sawflies by baculoviruses, in Microbial Control of Pests and Plant Diseases (Burges, H. D., ed.), Academic, London, pp Cunningham, J. C. (1988) Baculoviruses: their status compared to Bacilus thuringiensis as microbial insecticides. Outlook Agriculture 17, Young, S. Y., III and Yearian, W. C. (1982) Control of insect pests of agricultural importance by viral insecticides, in Microbial and Viral Pesticides (Kurstak, E., ed.), Marcel Dekker, New York, pp Huber, J. (1986) Use of baculoviruses in pest management programs, in The Biology of Baculoviruses, vol. II (Granados, R. R. and Federici, B., eds.), CRC, Boca Raton, FL, pp